ABSTRACT A major goal of oncolytic virotherapy is systemic delivery to metastatic disease. However, currently, i.v. virus cannot access tumors at sufficient levels to achieve regression(s). Therefore, novel protocols must be developed by which viruses can survive in the circulation long enough to access tumors in the face of anti viral neutralizing antibodies (NAb), components of the circulation which inactivate the viruses, and vascular barriers preventing extra-vasation. In our Phase I clinical trial of systemic delivery of Reovirus, there is encouraging evidence of virus reaching metastatic tumors. We will now return to our pre-clinical models, using Vesicular Stomatitis Virus (VSV), to treat B16 murine tumors in immune competent mice. To enhance virus survival in the circulation we will use cyclophosphamide (CPA), which suppresses anti-viral innate/adaptive responses and should be acceptable to regulatory authorities as an adjunct to systemic virotherapy. We have shown that, depending upon dose/timing of CPA, high levels of systemic virus can access s.c. tumors and both toxicity, and levels of NAb (which control access of the virus to systemic tissues), can be regulated. We will also target the major physical barrier of the tumor vasculature and have shown that induction of vascular permeability safely facilitates access of circulating virus into tumors along with significant therapy. Therefore, our overall hypothesis is that it will be possible to develop clinically applicable protocols by which oncolytic viruses can be delivered systemically to established tumors, at therapeutic levels, in a fully immune competent host. To test this hypothesis, we will optimize the tumor localization/replication of intravenous oncolytic virus following a first administration in an immune-competent host (Aim 1). In Specific Aims 2 and 3, we will optimize the tumor localization/replication of i.v. virus using repeat administrations by modifying the timing of administration, the nature of the virus (Aim 2) or the host immune system (Aim 3). Finally, we will combine the optimal conditions for systemic delivery from Aims 1-3 to treat well-established subcutaneous and metastatic disease (Aim 4). These experiments will drive the initiation of new trials of VSV as a systemic agent at the Mayo Clinic to complement our ongoing trials with other oncolytic viruses.